Carroll Vance at the University of Minnesota completed a functional comparison of root transcript expression between Clark (iron efficient) and Iso-Clark (iron inefficient) isogenic lines. Differential expression of genes belonging to specific functional pathways was plotted with the Mapman software program. He mapped several processes and pathways including: iron response metabolism, transport processes, receptor kinases, transcription factors, and regulatory processes.

NAS3 is a gene that encodes for a protein that synthesizes nicotianamine. Iron is attached to this molecule when it is transported through the phloem from older to young leaves and into the seed. To study this gene further, homozygous transgenic lines that over-expressing NAS3 have been developed, and the T2 seeds harvested. In collaboration with Dr. Robert Stupar, they will grow NAS3 lines and phenotype them. Last year they developed transgenic plants containing the Vit1 gene. Seed germination from the transformed Vit1 lines has been poor and appears to have no more viable plants. This year they will now focus on the NAS3

Robert Stupar has performed comparative genomic hybridization (CGH) analysis on iron-efficient and iron-inefficient isolines from the backgrounds Clark and IsoClark, respectively. The purpose of this experiment was to determine if there were any structural differences that could point to a genomic region that might contain genes that are related to iron metabolism. They tested seven different sub-lines from the IsoClark background using both the genotyping with the CGH technology and hydroponic screening to determine the IDC response of the lines. The hydroponic analysis was performed in collaboration with Dr. Randy Shoemaker, USDA/ARS, Ames, Iowa. They found unique genetic differences between the seven IsoClark sub-lines; however, the phenotypic assessment did not reveal any differences in IDC susceptibility. Despite the genetic polymorphisms between the IsoClark sub-lines, all seven lines were identical for the candidate gene Glyma03g28611. Therefore, the data offered further evidence that the major IsoClark IDC QTL may be conferred by this gene. This work has recently been accepted for publication (Stec et al., Front. Plant Sci. 4:104).

The Glyma03g28611 candidate gene is a ßHLH transcription factor that controls iron signaling. They are further investigating the function of our candidate gene. They have initiated transgenic studies to further investigate the function of this gene. They are developing gene constructs to transform this gene into the IsoClark background to test for improved IDC tolerance. Furthermore, they are also developing TALEN constructs to mutate this gene in two IDC tolerant lines (Clark and Bert). The TALEN system utilizes a zinc-finger nuclease to generate mutations in a target gene, in this case the Glyma03g28611 transcription factor. These mutants will allow the team to confirm the essential function of this gene. They plan to evaluate the transgenic lines for enhanced IDC tolerance when homozygous lines are mature enough to harvest enough seed to perform our intended experiments. These data should allow assessing the impact of this gene and this specific IsoClark mutation on the IDC trait.

Phillip McClean performed extensive genotyping of our association mapping (AM) population using the genotype-by-sequencing method to collect SNP data. For each line (n=280), he obtained on average 1,777,620 tags (~60 bp). A total of 79,000 SNPs were identified using the combined samples with the TASSEL pipeline. A total of 34,435 and 35,185 SNPs with a minor allele frequency >0.05 were discovered for the 2005 and 2006 mapping populations, respectively. For the combined data set, 34,428 SNP were discovered. The best AM model for the 2005 population utilized a principal component and kinship cofactors, while for the 2006 population the best model only included the kinship cofactor. A total of 281 SNP loci were significant with data combined over the two populations. Individually, 321 SNPs were significant in 2005 and 314 SNP in 2006. Significant genes within one megabase of the significant SNPs were identified.